1. Field of the Invention
The present invention relates to hybrid genes and enzymes of glucanase and dextransucrase, and to processes for preparing isomalto-oligosaccharides or dextran using the same. More specifically, the present invention relates to hybrid genes of glucanase and dextransucrase, recombinant vectors comprising said hybrid genes, microorganisms which are transformed with said recombinant vectors, hybrid enzymes which are expressed from said hybrid genes, and to process for preparing isomalto-oligosaccharides or dextran using said microorganisms or enzymes.
2. Description of the Prior Art
Recently, new kinds of sugar alternatives, “oligosaccharides,” that are derived from natural food sources, have been developed by biotechnological methods, and used to prevent problems associated with over-consumption of sugar and sugar derivatives, including dental caries, obesity, diabetes, adult diseases and the like (see, Kim, K. S. and Y. H. Chae, 1997, The effects of addition of oligosaccharide on the quality characteristics of tomato jam, Korean J. Food Sci 27(2): 170-175). Unlike typical sugars that are degraded and absorbed in the form of monosaccharides by digestive enzymes present in human body, oligosaccharides taken as food materials are not readily degraded by digestive enzymes, thus producing less calorie compared to sucrose (see, Kim, K. S and Y. H. Chae, 1997, The effects of addition of oligosaccharide on the quality characteristics of tomato jam, Korean J. Food Sci 27(2): 170-175). Further, oligosaccharides have probiotic and seedling effects by promoting the growth of probiotic bacteria, Bifidobacteria, an inhibitory effect on the increase of blood glucose or cholesterol level (see, Zakia, S. and C. Andrieux, 1997, Compared effects of three oligosaccharides on metabolism of intestinal microflora in rat inoculated with a human fecal flora, Br. J. Nutr. 78: 313-324), and an inhibitory effect on synthesis of glucan that causes dental caries (see, Kim, K. S and Y. H. Chae, 1997, The effects of addition of oligosaccharide on the quality characteristics of tomato jam, Korean J. Food Sci 27(2): 170-175). Oligosaccharides include soybean-oligosaccharide, fructo-oligosaccharide, galacto-oligosaccharide, isomalto-oligosaccharide, etc. Isomalto-oligosaccharide is comprised at a small amount in soybean paste, soy sauce and rice wine, etc. It has a chemical structure wherein two or three saccharide residues are linked each other, and each saccharide residue is composed of one to six glucose molecules. Isomalto-oligosaccharide is known as a seasoning for foods and has the sweetness of about 50% compared to that of sucrose. Meanwhile, dextran is a polymer of D-glucose, having a molecular weight of about 4 million Daltons in its natural state. Dextran is used as a raw material for preparing syrup, etc., and further, it can be partially hydrolyzed by an acid and dissolved in a physiological saline at a concentration of about 6% for use as a serum substitute. Dextran, which has a molecular weight of from 5,000 to 100,000 Daltons, including one for use as a serum substitute, is called as ‘clinical dextran.’
At present, various kinds of oligosaccharides are industrially produced either by the hydrolysis of polymers with an enzyme or an acid, or by the treatment of substrate with a glycosyl transferase. Moreover, for producing clinical dextran, a new method of mixed-culture fermentation has been developed. Compared to existing commercial methods for producing dextran that involve cultivation of microorganisms and acid-hydrolysis, the mixed-culture fermentation is simpler and gives a higher yield (see, Kim, D. and D. F. Day, 1994, A new process for the production of clinical dextran by mixed-culture fermentation of Lipomyces starkeyi and Leuconostoc mesenteroides, Enzyme Microb. Technol. 16: 844-848). The mixed-culture fermentation can be used to produce dextran with a desired low molecular weight by co-cultivating two kinds of bacteria, e.g., Lipomyces starkeyi which produces dextranase to hydrolyze dextran and Leuconostoc mesenteroides which produces dextransucrase to synthesize dextran, in a single fermentor. However, this method requires a delicate control for optimizing the growth of each bacterium and for suitably regulating the feed rate of sucrose.
Another method for producing oligosaccharides involves reacting sucrose substrate with dextransucrase and dextranase at the same time, thereby producing dextran by the action of dextransucrase and degrading the produced dextran by the action of endodextranase. When oligosaccharides are produced by using dextransucrase and dextranase, each strain producing each enzyme is cultivated separately, the resulting two enzymes are prepared separately, and then, the two enzymes are mixed in a ratio appropriate for obtaining a desired enzymatic activity or are prepared as immobilized enzymes for further use. However, this method requires a special mechanism for controlling the activities of said two enzymes.
Meanwhile, still another method involves immobilization of dextransucrase in various ways, reacting sucrose substrate with the immobilized dextransucrase, and then, degrading the thus obtained dextran with water-soluble dextranase. However, in the case of reacting the substrate with the two enzymes, some problems are occurred including that production yield of isomalto-oligosaccharides is significantly lower than the degradation rate of sucrose and production yield of polysaccharides are higher than that of isomalto-oligosaccharides. In addition, it requires a complicated technique for cultivating two strains separately and controlling activities of two enzymes simultaneously.
Under the circumstances, there has been a strong demand for a new method for simple and effective production of isomalto-oligosaccharides or low molecular weight dextran for clinical use from sucrose substrate by using a single bacterial strain or enzyme.